Cell bio midterm 3

Cell doctrine

Cell is going to duplicate itself and then divide into two different genetically identical cells

High fidelity cell division

The process of genome duplication and cell division is extremely high fidelity: little to no mistakes

Cell cycle stages

Interphase: G1, S, G2

Mitosis

G0

G1 phase

growth, monitoring: is DNA damaged?

S phase

DNA replication

G2 phase

monitoring

G0

non-dividing cells are in this stage

Cell cycle is evolutionarily conserved: yeast genetics

• Deleted the yeast Cdk gene: kills the cells because it stops the cells from growing

• put in human Cdk gene into the yeast: cells are alive again

• Yeast genome is haploid

• Yeast morphology makes it easy to see what stage of the cell cycle a cell is in

Temperature-sensitive yeast mutants

• permissive temperature (yeast cells grown at low temp): they are alive and growing, cells at every different cell cycle stage

  • protein is folded and functional

• non-permissive temperature (high temp): cdc proteins start becoming inactive, and mitosis stops

  • protein is not folded and non-functional

Fluorescence-activated cell (FAC) sorter

• use a fluorescent dye that can get into cells and bind to the DNA

  • we get an amount of dye trapped in the cell that is based on the amount of DNA in the cell

• The cells will go past the laser individually and the laser activates the fluorescent dye on the DNA and the detector detects how much fluorescence is in the cell and therefore how much DNA is in the cell

• Allows you to measure the amount of DNA in each cell as it passes through the laser

• The detector records the quantity of DNA in the cell

• SEE DRAWING 4/2

• G1: 1x DNA; G2: 2x DNA

• Mutants can have G2 arrest where they get stuck with 2x DNA (cant go back to G1) or

  • G1 arrest where they get stuck with 1x the DNA (so they cannot go to S phase)

Cell cycle control system

• Clock: keeps track of time, how much time in each cell cycle stage, determined when the cell should transition to the next stage

• Switches: the things that actually trigger the cell cycle transitions from G1 to S, G2 to mitosis, and metaphase to anaphase

• Mechanisms: things that monitor problems (eg. DNA damage)

  • at each transition there are checkpoints

  • End of G1 ( is there damaged DNA? if there is then the checkpoint stops the cell from transitioning)

  • End of G2 (any damaged DNA? if yes then no mitosis)

  • End of metaphase (is there a problem with how the chromosome is attached to the spindle? if yes then stop the cell from going to anaphase)

Cyclin-Cdk

• Clock of the cell cycle

• Dimer

  • Cdk is the kinase that phosphorylates substrates

  • Other protein is cyclin

• Kinase has zero activity unless it is bound to cyclin: the cyclin is essential for the kinase to have any activity

Cell fusion experiments (cells from different cell division stages)

• fuse a G2 cell and a mitotic cell: G2 cell immediately enters mitosis (because of M-Cdk activity from the mitotic cell): M-Cdk is dominant

• Fusion of S and G1 cells: The G1 cell enters S-phase (because of S-cyclin activity in the S phase cell): induces replication

Cyclin-dependent kinase

ALWAYS PRESENT

protein abundance is constant through the cell cycle

only active when bound to cyclin

Cyclin

• activates Cdk AND gives Cdk substrate specificity

• cyclin protein abundance oscillates through the cell cycle (dependent on transcription of the cyclin gene and the protein degradation of the cyclin protein (proteolysis)

M-cyclin

• M cyclin gives substrate specificity for lamin, etc.

• Gets made at the end of G@ (gene gets transcribed and translated so it can activate the Cdk)

• Gets degraded at the end of mitosis to G1 transition so you end up with just Cdk on its own again

• This limits the amount of time that M-cyclin is present to just mitosis

S-cyclin

• The S cyclin gives substrate specificity for DNA replication proteins

• Gets made at the end of G1

• Gets degraded towards the end of S phase

• When it gets degraded Cdk is free again

The structural basis/mechanism of Cdk activation by cyclins

• The T-loop (part of the 3D AA sequence of the Cdk) is obscuring the active site of Cdk

• When it binds cyclin, there is a conformation change that moves the T loop and exposes the active site, resulting in a partly active kinase

• for it to be fully activated, CAK must phosphorylate the T loop so that there is a further conformation change that moves the T loop even farther away from the active site to give full activation

• without cyclin there is no cdk activity even with CAK present

Ubiquitin dependent proteolysis/degradation

• Ub is a small protein that can get covalently attached to other proteins in chains by a ubiquitin ligase (APC/C)

• flags protein to get degraded by the proteasome: the only protease that will degrade these proteins when Ub is attached

Ubiquitin ligase (APC/C) (anaphase promoting complex/cyclosome)

• Present throughout the whole cell cycle

• gets activated and given substrate specificity at specific cell cycle transitions by activating subunits:

  • Cdh1: binds to the APC/C to give a complex that is an active ubiquitin ligase that ubiquitinates (causes degradation of) the M-cyclin (can also degrade S-cyclin in G1 if not inhibited)

  • Cdc20: APC/C-cdc20 ubiquitinates securin (typically keeps separase inactive, prevents transition from metaphase to anaphase)

Cell cycle transitions

G1 → S: S-cyclin activates cdk

G2 → M: M-cyclin activates cdk

Meta → Ana: cdc20 activates APC/C: degrades securin

M → G1: cdh1 activates APC/C: degrades M-cyclin

Regulating Cdk activity

• Wee1 kinase: adds inhibitory phosphate → completely inactivates the kinase

• Cdc25 phosphatase: takes off the inhibitory phosphate

• CKI-Cdk inhibitor proteins

  • p21 and p27 can inhibit S-Cdk

  • get degraded by ubiquitination by SCF ligase: always active

    • substrte has to get a phosphorylation on a particular residue for the SCF to be able to ubiquitinate it

Separase

• a protease that cleaves and degrades the cohesin protein to separate the sister chromatids and begin anaphase

• always present but inactive for most of cell cycle because it is otherwise bound to securin, which keeps the separase inactive

Cell cycle check points

• stop the cell cycle when something goes wrong

• G2 DNA damage checkpoint

  • check for damage (by x rays, etc)

  • If the cell detects damaged DNA, it activates Wee1 and inactivates Cdc25 to inhibit M-Cdk (blocks transition to metaphase)

• G1 DNA damage checkpoint

  • checks for damage

  • if damaged, activates p53 (transcription factor that binds to promoter of the p21 gene → p21 is made which inhibits S-Cdk)

• Spindle attachment checkpoint (SAC)

  • checks if all the chromosomes are bioriented

  • regulates metaphase → anaphase transition

  • SAC protein called Mad2 inhibits APC/C-Cdc20

Three main factors (ligands) that tell you the number and size of cells

• Mitogens: bind to the cell and stimulate cell division

  • in the absence of a mitogen, cells exit the cell cycle to G0

    • can be permanent or transient: do not have a mitogen bound but are capable of division if a mitogen does bind

• Growth factors

• Survival factors

G0 → G1

• Mitogen binds to a cell surface receptor

  • activates a signaling pathway: cell transcribes a gene that makes a protein called Myc

  • Myc activates expression of gene that encodes the G1 cyclin

  • G1 cyclin hooks up with Cdk to make G1-Cdk complex, which phosphorylates Rb

• E2F is activated which promotes S-cyclin to be made

  • When there is no mitogen, E2F is kept inactive by being in a complex with a protein called Rb

G1 → S

• Rb is phosphorylated by G1-Cdk so the E2F can promote the transcription of S-cyclin

• CKI proteins are phosphorylated by G1-Cdk to inhibit them

• Cdh1-APC/C is inhibited (phosphorylated by G1-Cdk) to prevent the S-cyclin from getting degraded

• SCF ubiquitinates CKI proteins (recognizes the phosphate added by G1-Cdk)

Substrates of the G1-Cdk

• Rb

• Cdh1-APC/C

• CKI proteins: p27

When does apoptosis occur?

• No survival factor: all cells must have a ligand (survival factor) bound to a receptor on the outside of the cell at all times for it to live

• Death factor (pro-apoptotic ligand) present: another ligand that can bind to the cell surface and can induce cell death

• Too much DNA damage:

  • a cell with some DNA damage activates p53 which activates p21 which causes G1 arrest for the cell to try to fix the damage

  • a cell with too much damage still activates p53, p53 this time activates BH3-only → induces apoptosis

Activation of apoptosis

• Intrinsic pathway: too much DNA damage, induced from within the cell when the cell detects too much DNA damage

• Extrinsic pathway: signal comes from outside the cell: a ligand provided by some other cell that tells the cell to go through apoptosis

• Caspases

  • all of our cells have caspases inside but all the time they are kept in an inactive state; must be activated by a signal

  • proteases that have a cysteine at their active site and cleave their target proteins at specific aspartic acids

  • Two types of caspases

    • initiator caspases

    • Executioner caspases

Caspases: general mechanism

• Called procaspases when inactive

  • monomers

  • for them to get activated they need to come together into a dimer or a bigger cluster of caspases

• Have a cysteine at their active site and cleave their target proteins at specific aspartic acids

• Pro domain and protease domain

• An adaptor protein has domains that bind to adaptor binding domain on the procaspase, recruits initiator caspases, initiator caspase cleave each other, subunits reorganized into a new complex: active caspase complex

  • bring the initiator procaspases together, cleave subunits, now active

• Similar rearrangement for activating executioner caspase

CAD

• Protein that chops genome into little pieces

• Normally CAD is bound to iCAD (inhibitor)

• executioner caspase cleaves the polypeptide backbone of iCAD, releases the now active CAD protein

Is the caspase cascade self-amplifying and irreversible?

yes

Extrinsic pathway

• Cell expresses fas death receptor (transmembrane protein) when it knows it has to be eliminated by apoptosis

• Killer lymphocytes have a Fas ligand that induce apoptosis by binding to Fas death receptor and causing activation and clustering of the receptors

• Each Fas receptor has an intracellular portion called a death domain

  • when the receptors cluster, these domains align, allowing the FADD adaptor protein to bind to the receptors

• FADD recruits initiator caspase called caspase8: domain binds to the adaptor

• Initiates caspase cascade that leads to apoptosis

Proteins that inhibit the extrinsic pathway

• Decoy receptors: have a ligand binding domain on the outside of the cell but do not have a death domain: not able to recruit the adaptor protein

• Intracellular proteins such as FLIP: structures like an initiator procaspase but does not have a proteolytic domain: non-functional and gets in the way of the real initiator complexes

Intrinsic pathway

• cytochrome c gets released from the mitochondria

• cytochrome c binds to adaptor protein in the cytoplasm: Apaf1

• conformation change that exposes two domains in the Apaf1 protein

  • oligomerization domain: allows it to bind to other Apaf1s

  • CARD domain: binds to initiator procaspase: caspase 9, which also have CARD domains that bind to the CARD domains of the Apaf1 oligomer

  • Cross cleavage occurs so initiator caspases are now active and can activate executioner caspases to activate apoptosis

Regulation of cytochrome c

• want to keep it in the mitochondria until it’s time for apoptosis

• Bclxl:

  • 4 domains

  • Blocks Bak-Bak oligomerization

  • Mimics the Bak protein but also has a BH4 domain

  • Has a BH3 binding groove that binds to the Bak protein, creating a heterodimer between Bclxl and Bak

    • these heterodimers cannot form the pore in the mitochondrial membrane

• Bak:

  • 3 domains

  • Activation: exposes a BH3 domain, and creates a BH3 binding groove → induces Bak-Bak oligomers → leads to cytochrome C release

  • Inactive Bak is a monomer

  • Forms oligomers in the mitochondrial membrane that forms pores in the membrane so that cytochrome C can diffuse out

  • When activated, the BH3 binding groove is exposed and can bind to the BH3 binding groove of another Bak protein to create on oligomer

• Bad:

  • only has a BH3 domain

  • inhibits Bclxl

  • p53 acts as a transcription factor that activates the Bad gene that induces apoptosis

Junction functions

• organize tissues

• mechanical support

• tissue permeability

• cell migration

Categories of junctions

• Anchoring junctions

  • Basically stick cells together or to the ECM (cell-cell junction or cell-ECM junction)

• Occluding junction

  • tight junction: cell-cell junction

  • tissue permeability

Occluding junctions between epithelial cells

• epithelial cells are stuck together by tight junctions

• forms a permeability barrier

• glucose molecules cannot pass between cells

• allows tissues to regulate glucose

• really tightly seal cells together

• multipass membrane proteins cluster together and interact with a similar protein next to it

Velcro principle of junctional complex

• Cadherins and integrins can form two broad types of junction

  • Junction

    • Weak interactions (transient)

    • Small number of cadherin molecules serving as the cell adhesion molecules

  • Junctional Complex

    • Could use the same cadherin to make a complex where many cadherins form a super strong complex

    • Many clustered interactions: Very strong

    • Velcro principle

Adherens junctions

• Cell-cell

• cadherin

• Adhesion belt of actin filaments circling the top of the cell inside

  • attached to the cadherins

  • adhesion belt is along a sheet of epithelial cells and tightens to make the sheet form a tube

Desmosomes

• where cells need to be attached to each other really really strongly

• Cadherins hold the junctions together

• Cadherins bind to the anchor proteins

• Anchor proteins are attached to intermediate filaments

Hemi-desmosomes

• like a desmosome but attaches cells to the ecm very strongly

• uses integrin instead of cadherin

• has anchor plaque that is attached to intermediate filaments

Focal adhesions

• integrin is the transmembrane protein

• anchor proteins interact with actin

• integrin binds to ECM protein (fibronectin or laminin)

• when the actin moves the cell, the actin is attached to integrins which is attached to the ECM: the integrins are kind of like feet that walk along the ECM

• Junction: weak and transient integrin-fibronectin binding: allows cell migration

• junctional complex: integrin attaches to a tendon, very strong connection

Cadherins

• There are a ton of different kinds of cadherins

• Experiment: took two different cell types that have different cadherins, incubate together, they move around and sort out: red binds to red, blue binds to blue

• Cells bind to each other when they have the same cadherin on the same surface because the cadherins bind to each other homotypically

• Embryogenesis: starts as a large bunch of cells but they can sort themselves out

  • All start out as one kind of cadherin, some start to switch to different type so can no longer attach to the originials, can migrate elsewhere and differentiate into different types of cells

  • cells change their behavior based on the type of cadherin they are expressing

• Cadherin cell-cell interactions dictate whether or not cells move

• calcium makes cadherin more rigid

  • in between the cadherin domains are hinge regions where the calcium binds and keeps the cadherin straight

    • important for allowing the cadherin to bind to the cell next to it

  • Calcium dependent binding (calcium ions bind to the hinge region between cadherin repeats): keeps the cadherin rigid— needed for cell-cell interactions

Cell migration

• cell-cell junctions dictate if a cell can migrate

• cell-ECM junctions dictate where a cell migrates

  • integrins follow particular binding sites (a trail that brings them to the right place)

Components of ECM

• Proteoglycans

• Adhesive proteins

  • collagen

  • fibronectin

  • laminin

• Water

• Few cells

  • fibroblasts (secrete the other things that make up the ECM)

Proteoglycans

• Protein + GAG

• A sugar is called a GAG that gets covalently attached to a protein in the Golgi (GAG is a big, long sugar polymer that proteins get attached to, brings a lot of water in and swells up inside the ECM, puts an outward pressure on the tissue/ECM)

• Main function of GAGs is to resist compression

  • The sugars have a lot of negatively charged parts to them

  • The sugar binds to a lot of water

    • Makes a hydrated gel: ECM withstands compression forces (aggrecan aggregate)

Collagen

• can do a bunch of things

  • branch, form cables

• Fibrillar collagen

  • Made of triple helix of alpha chains that bundle to form fibrils that bundle to form fiber

• Helps tissues withstand stretching, gives them tensile strength (tendons, ligaments, skin)

Collagen synthesis

1. Pro-alpha chains imported into the ER

2. Hydroxylation (OH group added) to prolines and lysines in the ER (OH groups hydrogen bond along the alpha chains to stabilize the triple helices)

3. Triple helices form (Golgi) and are stabilized by H bonding

4. Triple Helices secreted

5. In the ECM, a protease cuts off the very N terminal and very C terminal bit of the helix (the pro peptides) → leads to fibril formation

Fibronectin and laminin

• Adhesive proteins

• has binding sites for a bunch of different parts of the ECM

• allow the ECM to make this complex meshwork because it can bind to lots of different things

• integrin binds to the RGD sequence (arginine, glysine, aspartic acid) on adhesive proteins

• disintegrin also has RGD sequence and can compete with adhesive proteins for the integrin and therefore make a weaker cell-ECM junction rather than a junctional complex, and allows the cell to migrate

Integrin

• sculptor of the embryonic body plan

• tells cells where to go

• weak interactions between integrin and the ECM allow for migration

The basic components of signaling

• Signaling molecule: ligand

  • proteins

  • steroids

  • solvent gas

• Receptors: ligands bind to

  • cell surface receptor: ligand is large and hydrophilic (cannot diffuse)

  • Intracellular receptors: ligand is small and hydrophobic (actually able to diffuse across the membrane of the cell and then will do something in the cell by binding to an intracellular receptor)

• Intracellular signaling proteins: pass signaling along in a signaling cascade

• Effector protein: actually does something and changes the behavior of the cell in some way

• At each step you have something activating something else

Types of signaling

• Contact-dependent

  • The ligand is a transmembrane protein on a cell that binds to the cell surface receptor next to it

  • Ligand is not free and diffusible

• Paracrine

  • The signaling cell secreted the ligand

  • The ligand is diffusible and binds to a receptor in the target cell

• Synaptic

  • signaling molecule is released

  • binds to receptor in a target cell

  • the synapses and the target cell are super close together (have junctions holding them together)

• Endocrine

  • releases ligand (hormone)

  • gets in the bloodstream and goes all over the whole body

  • finds the target cell within the body

  • by releasing a signaling molecule into the bloodstream, it can affect many cells throughout the whole body

Synaptic vs endocrine signaling

• Synaptic

  • nerve cell → target cell

  • super fast

  • transient

  • ligand is concentrated

  • can have low affinity for the receptor

  • ligand is typically unstable

  • small number of target cells

• endocrine

  • relatively really slow because the signalling molecule has to go all through the blood stream

  • can be long lived effects

  • hormone is really dilute

  • have to have high affinity of ligand for receptor since the hormone is so dilute

  • ligand has high stability

  • many cell types are affected by the same ligand

Types of intracellular receptor

• Guanylyl cyclase

  • intracellular receptor

  • makes cGMP (second messenger) from GTP: “diffusibile”

  • acetylcholine activates NOS in epithelial cell, which causes nitric oxide to be made and diffuse into smooth muscle cell

  • Ligand is nitric oxide: binds to guanylyl cyclase in smooth muscle cell

  • cGMP is made, which causes the muscles to relax

• Transcription factors: steroid hormone

  • ligand binding causes the transcription factor to get activated

Types of cell surface receptors

• Ion channel-linked receptors

• G-protein coupled receptors

• enzyme-linked receptor

How is signaling regulated?

• Kinase

  • Phosphorylation turns things on

  • Phosphatases inactivate signals

• GTPase

  • if signaling is on, it is bound to GTP; if it gets hydrolyzed, it gets turned off

Molecular Binding Domains

• SH2, PTB (bind phospho-tyrosine)

• SH3 (bind short proline-rich sequences)

• PH (bind phosphorylated inositol phospholipids)

Integrator proteins

• where two different signaling events get integrated to do a common thing

• an integrator protein may have two residues phosphorylated by two different proteins caused by the activation of two different ligands

G-coupled protein receptors (GPCR)

• GPCR receptor (a 7-pass membrane protein part of a large family)

• Protein complex: trimeric G protein

  • Has alpha, beta, and gamma subunits

  • the alpha subunit is a GTPase

  • Activates an enzyme

  • Starts off in the inactive form with the alpha unit bound to GDP

• GPCR binds ligand

• induces recruitment of the trimeric G protein

• induces the GDP to come off the alpha subunit

• the active alpha subunit binds to GTP and is released from the GPCR and the beta gamma subunits

• alpha activates an enzyme

Types of alpha subunits

• Determines which enzyme gets activated

• G alpha s: activates adenylyl cyclase

• G alpha i: inhibits adenylyl cyclase

• G alpha q: activates phospholipase C beta (PLC-beta)

What turns the GPCR signaling off

• GAP: induces GTP hydrolysis

• GCR kinase: phosphorylates the GPCR: blocks the trimeric G protein from binding the GPCR

Adenylyl cyclase

• activated by G alpha s

• makes cAMP

• cAMP activates protein kinase A (PKA)

• cAMP binds to the PKA inhibitor, releasing an activated PKA

• in general, PKA phosphorylates transcription factors

• often the consequence of cAMP activation is to induce transcription in different genes

G alpha q: inositol phospholipid signaling

• G alpha q activates PLC-beta

• PIP2 (lipid in membrane) gets converted by PLC-beta into DAG and IP3

• IP3 is a second messenger that goes to calcium release channels to release calcium ions (also a second messenger) from the ER

Effects of calcium ions

• activates calmodulin

  • calmodulin activates NOS (nitric oxide synthase)

  • calmodulin activates CaM kinase (activates transcription factors)

Enzyme coupled receptors

• To activate the receptor, the ligand brings two monomers together so they both activate each other

  • each of the monomers phosphorylate each other to give an activated receptor

• one type is receptor tyrosine kinases (RTKs)

Receptor tyrosine kinases (RTKs)